The GATA-4/5/6 subfamily of transcription factors regulates various aspects of cardiogenesis, from cell specification through heart tube morphogenesis and valve formation. The hypothesis of this proposal is that the GATA-4/5/6 genes contribute to a combinatorial code that defines cellular and morphological phenotypes during multiple steps of cardiogenesis. It will be important to determine the relative contribution of each gene to cardiogenesis, and how their relative expression patterns are established by upstream regulatory programs. Innovative animal models are needed to overcome limitations to current progress. We propose to use Xenopus embryos and novel lines of transgenic zebrafish to understand the function and regulation of GATA factors during heart development. The following aims will test the specificity of function and regulatory mechanisms controlling GATA-4/5/6: 1. Define the specificity of function for GATA-4/5/6 during early cardiogenesis. The Xenopus system will be exploited to effectively inhibit expression of each gene alone or in combination, within specific germ layers of embryos, and during specific induction assays in explants. 2. Define the role of regulatory factors to establish and maintain the GATA-4 cardiogenic expression pattern. Novel lines of transgenic zebrafish were generated that express GFP under the control of GATA-4 regulatory sequences. Using a reverse genetic approach the transgenic system will be used to define the cis-elements that are required to target expression to specific domains of the normal GATA-4 pattem, and the role of T-box factors will be further investigated since T-box binding sites regulate activity of an already identified upstream enhancer. 3. Identify novel regulatory pathways that control expression of GATA-4 and GATA-6. The expression patterns for these key regulatory genes are overlapping but distinct and are likely therefore to be controlled by some common pathways, but also some that arc specific. Candidate pathways will be tested in the context of our reporter fish and a forward genetic screen will be used to identify genes that control expression of GATA-4, GATA-6, or both.